Human erythrocytes exhibit an unusual biconcave disc shape together with remarkable stability and deformability, all of which are necessary for their survival in the circulatory system. It is now well established that the unusual cell shape and membrane mechanical properties are due in large part to the presence of an extensive membrane skeleton, composed primarily of the proteins spectrin and actin, that lines the inner membrane surface and to specific bridging interactions between this membrane skeleton and intrinsic membrane proteins in the lipid bilayer. The spectrin-actin skeleton associates with the membrane bilayer via two types of contacts, one involving short actin protofilaments and protein 4.1 interacting with the cytoplasmic domain of glycophorin C, and the second involving the bridging protein ankyrinR and protein 4.2 interacting with the cytoplasmic domain of the anion exchange protein (AE1). This project seeks to characterize the structure of the complex of proteins that forms this second class of bridging interactions in human erythrocytes. This focus is dictated by the knowledge that alterations in this second class of interactions results in spherical erythrocytes with decreased cell size and increased fragility, a condition known clinically as hereditary spherocytosis (HS). HS is a spectrum of diseases, occurring in one family out of 2,000 to 3,000, that present clinically as varying degrees of hemolytic anemia resulting from hemolysis of the spherical erythrocytes when they traverse the microcirculation. Recent data have indicated that 15-20% of HS cases are attributable to AE1 defects and ~50% of HS cases result from ankyrin defects (4). A reduction in protein 4.2 binding to the membrane is also a common finding (5). Studies over the past three decades have led to the definition of the intrinsic and peripheral membrane proteins that assemble to stabilize the erythrocyte membrane (reviewed in (6,7)). A central player in the assembly of these proteins is the cytoplasmic domain of AE1 (also known as band 3). The cytoplasmic domain of band 3 (cdb3) has been hypothesized to serve as an organizing center for binding ankyrinR (8) and protein 4.2 (9) as well as other cytosolic proteins including GAPDH (10,11), aldolase (12), phosphofructokinase (13), hemoglobin (14), hemichromes (15), and p72syk (16). Figure 1 shows a schematic diagram of some of the key protein-protein interactions that have been elucidated.